Enantiomeric liquid chromatography (LC) separations have attracted great attention in the past few decades. Currently, over a hundred chiral stationary phases (CSPs) have been reported, and these CSPs are made by coating or bonding the chiral selectors to supports, usually silica gel supports. Interestingly, only a few types/classes of CSPs dominate the field of enantiomeric separations. Polysaccharide-based CSPs, macrocyclic antibiotic CSPs, and π complex CSPs are examples of dominant classes. And within each class of CSP there often are one or two dominate entities. Thus, despite the introduction of many chiral selectors/CSPs, only a few are used for the majority of separations. It has been said that in order for any new CSP to make a substantial impact, it must fulfill one or more of the following requirements: (a) broader applicability than existing CSPs, (b) superior separation properties for specific enantiomers (e.g., better selectivity, higher efficiency, more beneficial solvent compatibilities, shorter separation times, lower cost, enhanced ability to use supercritical fluid chromatography, etc.), or (c) fill an important separation niche where no other CSPs are operative.
Cyclofructans (CFs) are one of a relatively small group of macrocyclic oligosaccharides. Cyclodextrins are perhaps the best known member of this class of molecules. However, CFs are quite different in both their structure and behavior. CFs are cycloinulo-oligosaccharides consisting of six or more β-(2→1)-linked D-fructofuranose units. Each fructofuranose unit contains four stereogenic centers and three hydroxyl groups. A common shorthand nomenclature for these compounds is CF6, CF7, CF8, etc., where CFn denotes a cyclofructan having n fructose moieties (i.e., 6, 7, 8, etc.) in the cyclic oligomer.
Cyclofructans were first reported by Kawamura and Uchiyama in 1989. They can be produced via fermentation of inulin by at least two different strains of Bacillus circulans. The gene that produces the cycloinulo-oligosaccharide fructanotransferase enzyme (CF Tase) has been isolated, and its sequence determined and incorporated into the common yeast, Saccharomyces cerevisiae. Hence, the facile production of CFs is possible. The basic structure of CFs is shown in FIG. 1. From the x-ray crystal structure of CF6 it is known that the smaller CFs have no hydrophobic cavities as do cyclodextrins. Consequently, hydrophobic inclusion complexation, which plays an important role in the association of organic molecules with cyclodextrins, does not seem to be relevant for cyclofructans.
Instead, the pentose moieties (fructoses) of CFs form a propeller-like circumference around a crown ether core unit. For example, the crystal structure of CF6 reveals that six fructofuranose rings are arranged in a spiral or propeller fashion around the 18-crown-6 core, oriented either up or down toward the mean plane of the crown ether. Six three-position hydroxyl groups alternate to point toward or away from the molecular center, and the three oxygen atoms pointing inside are very close to each other (˜3 Å). It is clear that there is considerable internal hydrogen bonding in the cyclofructan molecule. As a result, access to the 18-crown-6 core on one side of the macrocycle is blocked by the hydrogen bonded hydroxyl groups. The other side of CF6 appears to be more hydrophobic, resulting from the methylene groups of —O—C—CH2—O— around the central indentation. A computational lipophilicity pattern of CF6 also confirms that CF6 shows a clear “front/back” regionalization of hydrophilic and hydrophobic surfaces. Both the crystal structure and computational modeling studies demonstrate that CF6 appears to have considerable additional internal hydrogen bonding. The fact that three 3-OH groups completely cover one side of the 18-crown-6 ring and the core crown oxygens are almost folded inside the molecule makes CF6 very different from other 18-crown-6 based chiral selectors. Table 1 gives relevant physico-chemical data for CF6, CF7 and CF8.
TABLE 1Physico-chemical properties of cyclofructans 6-8.Melting[α]D20 (°) inCavity I.D.MacrocycleMacrocycleMacrocycleM.W.Point (° C.)H2O(Å)O.D. (Å)Height (Å)CF6972.84  210-219a−64.62.314.68.7-9.4(231-233)CF71134.98215-222N/A4.115.98.5-8.9CF81297.12N/AN/A4.716.18.5-9.2aMelts and decomposes in this range.
Cyclofructans have been used in a variety of applications, mostly as an additive in consumer products or as a means to associate metal ions in solution. Many applications of CFs as additives in consumer products are similar to those of cyclodextrins. For example, CFs have been used as paper coatings, moderators of food and drink bitterness and astringency, browning preventive agents, emulsion polymerization agents, ink formulation agents, agents for suppressing smells, parts of drug delivery systems, lubricants, and so on. In addition, CFs have been shown to have cryoprotective effects and to be useful as complexing agents for some ions. However, to our knowledge CFs or their analogues have never been used as a broadly useful CSP for the separation of enantiomers by chromatography. Not intending to be bound by theory, it is possible that un-derivatized (or “native”) CFs cannot be used as CSPs for gas-liquid chromatography (GLC) due to their high melting points and their inability to solubilize in other liquid GLC stationary phases. For the above-mentioned structural reasons, it is also possible that native CF6 and other CFs have limited capabilities to form either hydrophobic inclusion complexes and/or crown ether inclusion complexes.